Flea synaptic vesicle nucleic acid molecules, proteins and uses thereof

ABSTRACT

The present invention relates to flea synaptic vesicle proteins; to flea synaptic vesicle nucleic acid molecules, including those that encode such flea synaptic vesicle proteins, respectively; to antibodies raised against such proteins; and to compounds that inhibit the activity of such proteins. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. The present invention also includes therapeutic compositions comprising such inhibitory compounds, particularly those that specifically inhibit flea synaptic vesicle activity, as well as the use of such therapeutic compositions to treat animals.

FIELD OF THE INVENTION

The present invention relates to flea synaptic vesicle nucleic acidmolecules, proteins encoded by such nucleic acid molecules, antibodiesraised against such proteins, and inhibitors of such proteins. Thepresent invention also includes methods to obtain such proteins, nucleicacid molecules, antibodies, and inhibitory compounds. The presentinvention also includes therapeutic compositions comprising suchinhibitors, as well as uses thereof.

BACKGROUND OF THE INVENTION

Flea infestation of animals is a health and economic concern for petowners. In particular the bites of fleas are a problem for animalsmaintained as pets because the infestation becomes a source of annoyancenot only for the pet but also for the pet owner who may find his or herhome generally contaminated with insects. Fleas also directly cause avariety of diseases, including allergy, and also carry a variety ofinfectious agents including, but not limited to, endoparasites (e.g.,nematodes, cestodes, trematodes and protozoa), bacteria and viruses. Assuch, fleas are a problem not only when they are on an animal but alsowhen they are in the general environment of the animal.

The medical importance of flea infestation has prompted the developmentof reagents capable of controlling flea infestation. Commonlyencountered methods to control flea infestation are generally focused onuse of insecticides, which are often unsuccessful for one or more of thefollowing reasons: (1) failure of owner compliance (frequentadministration is required), (2) behavioral or physiological intoleranceof the pet to the pesticide product or means of administration: and (3)the emergence of flea populations resistant to the prescribed dose ofpesticide.

Synaptic vesicle proteins, including SVP1 an SVP2 proteins of thepresent invention, have structural and sequence conservation with abacterial family of proton co-transporters, with the mammalianproton/glucose transporter, and with organic ion transporters. SVP has12 putative transmembrane regions that arise from an internalduplication. In mammals, SVP proteins are located on neural andendocrine vesicles and are thought to function in the uptake ofneurotransmitters into vesicles utilizing the proton gradient.Neurotransmitters in turn regulate the activity of the ion channels onthese membranes. In the Malpighian tubules, the activity of tile ionchannels determines the rate of diuresis, or fluid secretion from thehemolymph into tile lumen. Thus, inhibiting the transport ofneurotransmitters in tile HMT tissues may have significant effects ontile functions of these tissues. As such, flea SVP1 and a SVP2 proteinsof the present invention represent novel targets for anti-flea vaccinesand chemotherapeutic drugs.

Therefore, isolation and sequencing of flea SVP1 and SVP2 genes may becritical for use in identifying specific agents for treating animals forflea infestation.

SUMMARY OF THE INVENTION

The present invention provides flea synaptic vesicle nucleic acidmolecules; proteins encoded by such nucleic acid molecules; antibodiesraised against such proteins (i.e., anti-flea synaptic vesicleantibodies); mimetopes of such proteins or antibodies; and compoundsthat inhibit flea synaptic vesicle activity (i.e. inhibitory compoundsor inhibitors).

The present invention also includes methods to obtain such proteins,mimetopes, nucleic acid molecules, antibodies and inhibitory compounds.The present invention also includes the use of proteins and antibodiesto identify such inhibitory compounds as well as assay kits to identifysuch inhibitory compounds. Also included in the present invention aretherapeutic compositions comprising proteins, mimetopes, nucleic acidmolecules, antibodies and inhibitory compounds of the present inventionincluding therapeutic compounds derived from a protein of the presentinvention that inhibit the activity of flea synaptic vesicle proteins;also included are uses of such therapeutic compounds.

One embodiment of the present invention is an isolated flea synapticvesicle nucleic acid molecule that hybridizes with a nucleic acidsequence having SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8. SEQ ID NO:10, SEQ ID NO:11, and/orSEQ ID NO:12, under conditions that allow less than or equal to 30% basepair mismatch. Another embodiment of tile present invention is anisolated flea synaptic vesicle nucleic acid molecule having a nucleicacid sequence that is at least 70% identical to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:11, and/or SEQ ID NO:12 and fragments of suchproteins at least 10 amino acid residues in length.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include a nucleic acid molecule ofthe present invention. Also included are methods to produce such nucleicacid molecules, recombinant molecules, recombinant viruses andrecombinant cells.

Another embodiment of the present invention includes an isolated fleasynaptic vesicle protein that is at least 70% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:3 and/or SEQ IDNO:9 and fragments thereof at least 10 amino acid residues in length,wherein such fragments can elicit an immune response against respectiveflea synaptic vesicle proteins or have activity comparable to respectiveflea synaptic vesicle proteins.

Another embodiment of the present invention includes an isolated fleasynaptic vesicle protein encoded by a nucleic acid molecule thathybridizes with a nucleic acid sequence having SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:10, and/or SEQ ID NO:12, under conditions that allow less thanor equal to 30% base pair mismatch.

Another embodiment of the present invention includes a compositioncomprising an excipient and a compound selected from the groupconsisting of nucleic acid molecules, proteins, and antibodies of thepresent invention and a method to treat an animal for flea infestationcomprising administering such a composition to such an animal.

Another embodiment of the present invention includes a method to detectan inhibitor of flea synaptic vesicle activity, said method comprising(a) contacting an isolated flea synaptic vesicle protein of the presentinvention, with a putative inhibitory compound under conditions inwhich, in the absence of said compound, said protein has flea synapticvesicle protein activity, and (b) determining if said putativeinhibitory compound inhibits flea synaptic vesicle protein activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for flea synaptic vesicle nucleic acidmolecules, proteins encoded by such nucleic acid molecules, antibodiesraised against such proteins, and inhibitors of such proteins. As usedherein, flea synaptic vesicle nucleic acid molecules and proteinsencoded by such nucleic acid molecules are also referred to as synapticvesicle nucleic acid molecules and proteins, or SVP nucleic acidmolecules and SVP proteins, respectively. Flea synaptic vesicle nucleicacid molecules and proteins of the present invention can be isolatedfrom a flea or prepared recombinantly or synthetically. Flea synapticvesicle nucleic acid molecules of the present invention can be RNA orDNA, or modified forms thereof, and can be double-stranded orsingle-stranded; examples of nucleic acid molecules include, but are notlimited to, complementary DNA (cDNA) molecules, genomic DNA molecules,synthetic DNA molecules, DNA molecules which are specific tags formessenger RNA, and corresponding mRNA molecules. As such, a flea nucleicacid molecule of the present invention is not intended refer to anentire chromosome within which such a nucleic acid molecule iscontained, however, a flea SVP cDNA of the present invention may includeall regions such as regulatory regions that control production of fleaSVP proteins encoded by such a cDNA (such as, but not limited to,transcription, translation or post-translation control regions) as wellas the coding region itself, and any introns or non-translated codingregions. As used herein, the phrase “flea synaptic vesicle protein”refers to a protein encoded by a flea synaptic vesicle nucleic acidmolecule.

Flea synaptic vesicle nucleic acid molecules of known length isolatedfrom a flea, such as Ctenocephalides felis are denoted “nCfSVP1₈”, forexample nCfSVP1₁₈₇₅, wherein “#” refers to the number of nucleotides inthat molecule, and flea synaptic vesicle proteins of known length aredenoted “PCfSVP1₈” (for example PCfSVP1₅₃₀) wherein “#” refers to thenumber of amino acid residues in that molecule.

The present invention also provides for flea synaptic vesicle DNAmolecules that are specific tags for messenger RNA molecules. Such DNAmolecules call correspond to an entire or partial sequence of amessenger RNA, and therefore, a DNA molecule corresponding to such amessenger RNA molecule (i.e. a cDNA molecule), can encode a full-lengthor partial-length protein. A nucleic acid molecule encoding apartial-length protein can be used directly as a probe or indirectly togenerate primers to identify and/or isolate a cDNA nucleic acid moleculeencoding a corresponding, or structurally related, full-length protein.Such a partial cDNA nucleic acid molecule can also be used in a similarmanner to identify a genomic nucleic acid molecule, such as a nucleicacid molecule that contains the complete gene including regulatoryregions, exons and introns. Methods for using partial flea synapticvesicle cDNA molecules and sequences to isolate full-length andcorresponding cDNA molecules are described in the examples herein below.

The proteins and nucleic acid molecules of the present invention can beobtained from their natural source, or can be produced using, forexample, recombinant nucleic acid technology or chemical synthesis. Alsoincluded in the present invention is the use of these proteins andnucleic acid molecules as well as antibodies and inhibitory compoundsthereto as therapeutic compositions to protect animals from fleainfestation, as well as in other applications, such as those disclosedbelow.

One embodiment of the present invention is an isolated protein thatincludes a flea synaptic vesicle protein. It is to be noted that theterm “a” or “an” entity refers to one or more of that entity; forexample, a protein, a nucleic acid molecule, an antibody and atherapeutic composition refers to “one or more” or “at least one”protein, nucleic acid molecule, antibody and therapeutic compositionrespectively. As such, the terms “a” (or “an”), “one or more” and “atleast one” can be used interchangeably herein. It is also to be notedthat the terms “comprising”, “including”, and “having” can be usedinterchangeably. According to the present invention, an isolated, orbiologically pure, protein, is a protein that has been removed from itsnatural milieu. As such, “isolated” and “biologically pure” do notnecessarily reflect the extent to which the protein has been purified.An isolated protein of the present invention can be obtained from itsnatural source, can be produced using recombinant DNA technology, or canbe produced by chemical synthesis.

As used herein, isolated flea synaptic vesicle proteins of the presentinvention can be full-length proteins or any homologue of such proteins.An isolated protein of the present invention, including a homologue, canbe identified in a straight-forward manner by tile protein's ability toelicit an immune response against a flea synaptic vesicle protein or bythe protein's ability to exhibit flea synaptic vesicle activity.Examples of flea synaptic vesicle homologue proteins include fleasynaptic vesicle proteins in which amino acids have been deleted (e.g. atruncated version of the protein, such as a peptide), inserted,inverted, substituted and/or derivatized (e.g., by glycosylation,phosphorylation, acetylation, myristoylation, prenylation,palmitoylation, amidation and/or addition of glycerophosphatidylinositol) such that the homologue includes at least one epitope capableof eliciting an immune response against a flea synaptic vesicle protein,and/or of binding to an antibody directed against a flea synapticvesicle protein. That is, when the homologue is administered to ananimal as an immunogen, using techniques known to those skilled in theart, the animal will produce an immune response against at least oneepitope of a natural flea synaptic vesicle protein. The ability of aprotein to effect an immune response call be measured using techniquesknown to those skilled in the art. As used herein, tile term “epitope”refers to the smallest portion of a protein or other antigen capable ofselectively binding to the antigen binding site of an antibody or a Tcell receptor. It is well accepted by those skilled in the art that theminimal size of a protein epitope is about four to six amino acids. Asis appreciated by those skilled in the art, an epitope can include aminoacids that naturally are contiguous to each other as well as amino acidsthat, due to the tertiary structure of the natural protein, ale insufficiently close proximity to form an epitope. According to thepresent invention, an epitope includes a portion of a protein comprisingat least 4 amino acids, at least 5 amino acids, at least 6 amino acids,at least 10 amino acids, at least 15 amino acids, at least 20 aminoacids, at least 25 amino acids, at least 30 amino acids, at least 35amino acids, at least 40 amino acids or at least 50 amino acids inlength.

In one embodiment of the present invention a flea synaptic vesiclehomologue protein has flea synaptic vesicle activity, i.e. the homologueexhibits all activity similar to its natural counterpart. Methods todetect and measure such activities ale known to those skilled in theart.

Flea synaptic vesicle homologue proteins can be the result of naturalallelic variation or natural mutation. Flea synaptic vesicle proteinhomologues of the present invention can also be produced usingtechniques known in the art including, but not limited to, directmodifications to the protein or modifications to the gene encoding theprotein using, for example, classic or recombinant DNA techniques toeffect random or targeted mutagenesis.

Flea synaptic vesicle proteins of the present invention are encoded byflea synaptic vesicle nucleic acid molecules. As used herein, fleasynaptic vesicle nucleic acid molecules include nucleic acid sequencesrelated to natural flea synaptic vesicle genes, and, preferably, to C.felis flea synaptic vesicle genes. As used herein, flea synaptic vesiclegenes include all regions such as regulatory regions that controlproduction of flea synaptic vesicle proteins encoded by such genes (suchas, but not limited to, transcription, translation or post-translationcontrol regions) as well as the coding region itself, and any introns ornon-translated coding regions. As used herein, a nucleic acid moleculethat “includes” or “comprises” a sequence may include that sequence inone contiguous array, or may include the sequence as fragmented exonssuch as is often found for a flea gene. As used herein, the term “codingregion” refers to a continuous linear array of nucleotides thattranslates into a protein. A full-length coding region is that codingregion that is translated into a full-length, i.e., a complete proteinas would be initially translated in its natural millieu, prior to anypost-translational modifications.

One embodiment of the present invention is a C. felis flea synapticvesicle gene that includes the nucleic acid sequence SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:11, and/or SEQ ID NO:12. These nucleic acidsequences are further described herein. For example, nucleic acidsequence SEQ ID NO:2 represents the deduced sequence of the codingstrand of a C. felis cDNA denoted herein as C. felis synaptic vesiclenucleic acid molecule nCfSVP1₁₈₇₅, the production of which is disclosedin the Examples. Nucleic acid molecule SEQ ID NO:2 comprises anapparently full-length coding region. The complement of SEQ ID NO:2(represented herein by SEQ ID NO:4) refers to the nucleic acid sequenceof the strand fully complementary to the strand halving SEQ ID NO:2,which can easily be determined by those skilled in the art. Likewise, anucleic acid sequence complement of any nucleic acid sequence of thepresent invention refers to the nucleic acid sequence of the nucleicacid strand that is fully complementary to (i.e. can form a completedouble helix with) the strand for which the sequence is cited. It shouldbe noted that since nucleic acid sequencing technology is not entirelyerror-free. SEQ ID NO:2 (as well as other nucleic acid and proteinsequences presented herein) represents an apparent nucleic acid sequenceof the nucleic acid molecule encoding a flea synaptic vesicle protein ofthe present invention.

Translation of SEQ ID NO:2, the coding strand of nCfSVP1₁₈₇₅, as well astranslation of SEQ ID NO:5, the coding strand of nCfSVP1₁₅₉₀, whichrepresents the coding region of nCfSVP1₁₈₇₅, yields a protein of 530amino acids, denoted herein as PCfSVP1₅₃₀, the amino acid sequence ofwhich is presented in SEQ ID NO:3, assuming (a) an initiation codonextending from nucleotide 44 to 46 of SEQ ID NO:2, or from nucleotide 1to nucleotide 3 of SEQ ID NO:5, respectively; and (b) a stop codonspanning from nucleotide 1634 to 1636 of SEQ ID NO:2.

Translation of SEQ ID NO:8, the coding strand of nCfSVP2₃₃₁₄, as well astranslation of SEQ ID NO:11, the coding strand of nCfSVP2₂₃₁₉, whichrepresents the coding region of nCfSVP2₃₃₁₄, yields a protein of 773amino acids, denoted herein as PCfSVP2₇₇₃, the amino acid sequence ofwhich is presented in SEQ ID NO:9, assuming (a) an initiation codonextending from nucleotide 302-304 of SEQ ID NO:8, or from nucleotide 1to nucleotide 3 of SEQ ID NO:11, respectively; and (b) a stop codonspanning from nucleotide 2621 to 2623 of SEQ ID NO:8.

In one embodiment, a gene or other nucleic acid molecule of the presentinvention can be an allelic variant that includes a similar but notidentical sequence to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10. SEQ ID NO:11,and/or SEQ ID NO:12. For example, an allelic variant of a C. felissynaptic vesicle gene including SEQ ID NO:1, SEQ ID NO:2, SEQ LD NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:11, and/or SEQ ID NO:12 is a gene that occurs at essentially the samelocus (or loci) in the genome as the gene including SEQ ID NO:1, SEQ IDNO:2. SEQ ID NO:4. SEQ ID NO:5. SEQ ID NO:6. SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:11, and/or SEQ ID NO:12, but which, due tonatural variations caused by, for example, mutation or recombination,has a similar but not identical sequence. Because natural selectiontypically selects against alterations that affect function, allelicvariants (i.e. alleles corresponding to, or of, cited nucleic acidsequences) usually encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared.Allelic variants of genes or nucleic acid molecules can also comprisealterations in tile 5′ or 3′ untranslated regions of the gene (e.g., inregulatory control regions), or can involve alternative splicing of anascent transcript, thereby bringing alternative exons intojuxtaposition. Allelic variants are well known to those skilled in theart and would be expected to occur naturally within a given fleaspecies, since the genome is diploid, and sexual reproduction willresult in the reassortment of alleles.

In one embodiment of the present invention, isolated flea synapticvesicle proteins are encoded by nucleic acid molecules that hybridizeunder stringent hybridization conditions to genes or other nucleic acidmolecules encoding flea synaptic vesicle proteins, respectively. Theminimal size of flea synaptic vesicle proteins of the present inventionis a size sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid (i.e., hybridizing under stringent hybridizationconditions) with the complementary sequence of a nucleic acid moleculeencoding the corresponding natural protein. The size of a nucleic acidmolecule encoding such a protein is dependent on the nucleic acidcomposition and the percent homology between the flea synaptic vesiclenucleic acid molecule and the complementary nucleic acid sequence. I caneasily be understood that the extent of homology required to form astable hybrid under stringent conditions can vary depending on whetherthe homologous sequences are interspersed throughout a given nucleicacid molecule or are clustered (i.e., localized) in distinct regions ona given nucleic acid molecule.

The minimal size of a nucleic acid molecule capable of forming a stablehybrid with a gene encoding a flea synaptic vesicle protein is at leastabout 12 to about 15 nucleotides in length if the nucleic acid moleculeis CC-rich and at least about 15 to about 17 bases in length if it isAT-rich. The minimal size of a nucleic acid molecule used to encode aflea synaptic vesicle protein homologue of the present invention is fromabout 12 to about 18 nucleotides in length. Thus the minimal size offlea synaptic vesicle protein homologues of the present invention isfrom about 4 to about 6 amino acids in length. There is no limit, otherthan a practical limit, on the maximal size of a nucleic acid moleculeencoding a flea synaptic vesicle protein of the present inventionbecause a nucleic acid molecule of the present invention can include aportion of a gene or cDNA or RNA, an entire gene or cDNA or RNA, ormultiple genes or cDNA or RNA. The preferred size of a protein encodedby a nucleic acid molecule of the present invention depends on whether afull-length, fusion, multivalent, or functional portion of such aprotein is desired.

Stringent hybridization conditions are determined based on definedphysical properties of the flea synaptic vesicle nucleic acid moleculeto which the nucleic acid molecule is being hybridized, and can bedefined mathematically. Stringent hybridization conditions are thoseexperimental parameters that allow an individual skilled in the art toidentify significant similarities between heterologous nucleic acidmolecules. These conditions are well known to those skilled in the art.See, for example, Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al.,1984 , Anal, Biochem. 138, 267-284. As explained in detail in the citedreferences, the determination of hybridization conditions involves themanipulation of a set of variables including the ionic strength (M, inmoles/liter), the hybridization temperature (° C.), the concentration ofnucleic acid helix destabilizing agents (such as formamide), the averagelength of the shortest hybrid duplex (n), and the percent G+Ccomposition of the fragment to which an unknown nucleic acid molecule isbeing hybridized. For nucleic acid molecules of at least about 150nucleotides, these variables are inserted into a standard mathematicalformula to calculate the melting temperature, T_(m), of a given nucleicacid molecule. As defined in the formula below, T_(m) is the temperatureat which two complementary nucleic acid molecule strands willdisassociate, assuming 100% complementarity between the two strands:T _(m)=81.5° C.+16.6 log M+0.41 (% G+C)−500/n−0.61 (% formamide).For nucleic acid molecules smaller than about 50 nucleotides, hybridstability is defined by the dissociation temperature (T_(d)), which isdefined as the temperature at which 50% of the duplexes dissociate. Forthese smaller molecules, the stability at a standard ionic strength isdefined by the following equation:T _(d)=4(G+C)+2(A+T).A temperature 5° C. below T_(d) is used to detect hybridization betweenperfectly matched molecules.

Also well known to those skilled in the art is how base pair mismatch,i.e. differences between two nucleic acid molecules being compared,including non-complementarity of bases at a given location, and gaps dueto insertion or deletion of one or more bases at a given location oneither of the nucleic acid molecules being compared, will affect T_(m)or T_(d) for nucleic acid molecules of different sizes. For example,T_(m) decreases about 1° C. for each 1% of mismatched base pairs forhybrids greater than about 150 bp, and T_(d) decreases about 5° C. Foreach mismatched base pair for hybrids below about 50 bp. Conditions forhybrids between about 50 and about 150 base pairs can be determinedempirically and without undue experimentation using standard laboratoryprocedures well known to those skilled in the art. These simpleprocedures allow one skilled in the art to set the hybridizationconditions (by altering, for example, the salt concentration, theconcentration of helix destabilizing agents, or the temperature) so thatonly nucleic acid hybrids with greater than a specified % base pairmismatch will hybridize. Because one skilled in the art can easilydetermine whether a given nucleic acid molecule to be tested is lessthan or greater than about 50 nucleotides, and can therefore choose theappropriate formula for determining hybridization conditions, he or shecan determine whether the nucleic acid molecule will hybridize with agiven gene under conditions designed to allow a desired amount of basepair mismatch.

Hybridization reactions are often carried out by attaching the nucleicacid molecule to be hybridized to a solid support such as a membrane,and then hybridizing with a labeled nucleic acid molecule, typicallyreferred to as a probe, suspended in a hybridization solution. Examplesof common hybridization reaction techniques include, but are not limitedto, the well-known Southern and northern blotting procedures. Typically,the actual hybridization reaction is done under non-stringentconditions. i.e. at a lower temperature and/or a higher saltconcentration, and then high stringency is achieved by washing themembrane ill a solution with a higher temperature and/or lower saltconcentration in order to achieve the desired stringency.

For example, if the skilled artisan wished to identify a nucleic acidmolecule that hybridizes under conditions that would allow less thin orequal to 30% pail mismatch with a flea synaptic vesicle nucleic acidmolecule of about 150 bp in length or greater, tile following conditionscould preferably be used. Tile average G+C content of flea DNA is about37%, as calculated from known flea nucleic acid sequences. The unknownnucleic acid molecules would be attached to a support membrane, and the150 bp probe would be labeled, e.g. with a radioactive tag. Thehybridization reaction could be carried out in a solution comprising2×SSC in the absence of nucleic acid helix destabilizing compounds, at atemperature of about 37° C. (low stringency conditions). Solutions ofdiffering concentrations of SSC can be made by one of skill in the artby diluting a stock solution of 20×SSC (175.3 gram NaCl and about 88.2gram sodium citrate in 1 liter of water, pH 7) to obtain the desiredconcentration of SSC. The skilled artisan would calculate the washingconditions required to allow up to 30% base pair mismatch. For example,in a wash solution comprising 1×SSC in the absence of nucleic acid helixdestabilizing compounds, the T_(m) of perfect hybrids would be about 77°C.:81.5° C.+16.6 log (0.15M)+(0.41×73)−(500/150)−(0.61×0)=77.5° C.Thus, to achieve hybridization with nucleic acid molecules having about30% base pair mismatch, hybridization washes would be carried out at atemperature of less than or equal to 47.5° C. It is thus within tileskill of one in the art to calculate additional hybridizationtemperatures based on the desired percentage base pair mismatch,formulae and G/C content disclosed herein. For example, it isappreciated by one skilled in the art that as the nucleic acid moleculeto be tested for hybridization against nucleic acid molecules of thepresent invention having sequences specified herein becomes longer than150 nucleotides, the T_(m) for a hybridization reaction allowing up to30% base pair mismatch will not vary significantly from 47.5° C.

Furthermore, it is known in the art that there ale commerciallyavailable computer programs for determining the degree of similaritybetween two nucleic acid or protein sequences. These computer programsinclude various known methods to determine the percentage identity andthe number and length of gaps between hybrid nucleic acid molecules orproteins. Preferred methods to determine the percent identity amongamino acid sequences and also among nucleic acid sequences includeanalysis using one or more of the commercially available computerprograms designed to compare and analyze nucleic acid or amino acidsequences. These computer programs include, but are not limited to, theSeqLab® Wisconsin Package™ Version 10.0-UNIX sequence analysis software,available from Genetics Computer Group, Madison, Wis. (hereinafter“SeqLab”), and DNAsis® sequence analysis software, version 2.0,available from Hitachi Software, San Bruno, Calif. (hereinafter“DNAsis”). Such software programs represent a collection of algorithmspaired with a graphical user interface for using the algorithms. TheDNAsis and SeqLab software, for example, employ a particular algorithm,the Needleman-Wunsch algorithm to perform pair-wise comparisons betweentwo sequences to yield a percentage identity score, see Needleman, S. B.and Wunch, C. D., 1970, J. Mol. Biol., 48, 443. Such algorithms,including the Needleman-Wunsch algorithm, are commonly used by thoseskilled in the nucleic acid and amino acid sequencing art to comparesequences. A preferred method to determine percent identity among aminoacid sequences and also among nucleic acid sequences includes using theNeedleman-Wunsch algorithm, available in the SeqLab software, using thePairwise Comparison/Gap function with the nwsgapdna.cmp scoring matrix,the gap creation penalty and the gap extension penalties set at defaultvalues, and the gap shift limits set at maximum (hereinafter referred toas “SeqLab default parameters”). An additional preferred method todetermine percent identity among amino acid sequences and also amongnucleic acid sequences includes using the Higgins-Sharp algorithm,available in the DNAsis software, with the gap penalty set at 5, thenumber of top diagonals set at 5, the fixed gap penalty set at 10, thek-tuple set at 2, the window size set at 5, and the floating gap penaltyset at 10. A particularly preferred method to determine percent identityamong amino acid sequences and also among nucleic acid sequencesincludes using the Needleman-Wunsch algorithm available in the SeqLabsoftware, using the SeqLab default parameters.

One embodiment of the present invention includes a flea synaptic vesicleprotein. A preferred flea synaptic vesicle protein includes a proteinencoded by a nucleic acid molecule that hybridizes under conditions thatpreferably allow less thin or equal to 30% base pair mismatch,preferably under conditions that allow less than or equal to 20% basepair mismatch, preferably under conditions that allow less than or equalto 10% base pair mismatch, preferably under conditions that allow lessthan or equal to 8% base pair mismatch, preferably under conditions thatallow less than or equal to 5% base pair mismatch or preferably underconditions that allow less than or equal to 2% base pair mismatch with anucleic acid molecule selected from the group consisting of SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:10, and/or SEQ ID NO:12.

Another embodiment of the present invention includes a flea synapticvesicle protein encoded by a nucleic acid molecule that hybridizes underconditions comprising, (a) hybridizing in a solution comprising 1×SSC inthe absence of nucleic acid helix destabilizing compounds, at atemperature of 37° C. and (b) washing in a solution comprising 1×SSC inthe absence of nucleic acid helix destabilizing compounds, at atemperature of 47° C., to an isolated nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10,and/or SEQ ID NO:12.

Another preferred flea synaptic vesicle protein of the present inventionincludes a protein that is encoded by a nucleic acid molecule that ispreferably at least 70% identical, preferably at least 80% identical,preferably at least 90% identical, preferably at least 92% identical,preferably at least 95% identical or preferably at least 98% identicalto a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:11;also preferred are fragments (i.e. portions) of such proteins encoded bynucleic acid molecules that are at least 30nucleotides. Percent identityas used herein is determined using the Needleman-Wunsch algorithm,available in the SeqLab software using default parameters.

Additional preferred flea synaptic vesicle proteins of the presentinvention include proteins having the amino acid sequence SEQ ID NO:3and/or SEQ ID NO:9, and proteins comprising homologues of a proteinhaving the albino acid sequence SEQ ID NO:3 aid/or SEQ ID NO:9, whereinsuch a homologue comprises at least one epitope that elicits an immuneresponse against a protein having an amino acid sequence SEQ ID NO:3and/or SEQ ID NO:9. Likewise, also preferred are proteins encoded bynucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1, SEQID NO:2, SEQ ID NO:5. SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:11, orby homologues thereof.

A preferred isolated flea synaptic vesicle protein of the presentinvention is a protein encoded by at least one of the following nucleicacid molecules: nCfSVP1₁₈₇₅, nCfSVP1₁₅₉₀, nCfSVP2₃₃₁₄, and/ornCfSVP2₂₃₁₉, or allelic variants of any of these nucleic acid molecules.Also preferred is an isolated protein encoded by a nucleic acid moleculehaving nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:8, and/or SEQ ID NO:11; or a protein encoded by anallelic variant of any of these listed nucleic acid molecules.

Preferred flea synaptic vesicle proteins of the present inventioninclude proteins having amino acid sequences that are at least 70%,preferably 80%, preferably 90%, preferably 92%, preferably 95%,preferably at least 98%, preferably at least 99%, or preferably 100%identical to amino acid sequence SEQ ID NO:3 and/or SEQ ID NO:9 andproteins encoded by allelic variants of nucleic acid molecules encodingflea synaptic vesicle proteins having amino acid sequences SEQ ID NO:3and/or SEQ ID NO:9. Also preferred are fragments thereof having at least10 amino acid residues.

In one embodiment of the present invention, C. felis synaptic vesicleproteins comprise amino acid sequence SEQ ID NO:3 and/or SEQ ID NO:9(including, but not limited to, the proteins consisting of amino acidsequence SEQ ID NO:3 and/or SEQ ID NO:9, fusion proteins and multivalentproteins), and proteins encoded by allelic variants of nucleic acidmolecules encoding proteins having amino acid sequence SEQ ID NO:3and/or SEQ ID NO:9.

In one embodiment, a preferred flea synaptic vesicle protein comprisesan amino acid sequence of at least 6 amino acids, preferably at least 10amino acids, preferably at least 15 amino acids, preferably at least 20amino acids, preferably at least 25 amino acids, preferably at least 30amino acids, preferably at least 35 amino acids, preferably at least 40amino acids, preferably at least 50 amino acids, preferably at least 75amino acids, preferably at least 100 amino acids, preferably at least125 amino acids, preferably at least 150 amino acids, preferably atleast 175 amino acids, preferably at least 200 amino acids, preferablyat least 250 amino acids, preferably at least 300 amino acids,preferably at least 350 amino acids, preferably at least 400 aminoacids, preferably at least 450 amino acids, preferably at least 500amino acids, preferably at least 550 amino acids, preferably at least600 amino acids, preferably at least 650 amino acids, preferably atleast 700 amino acids, preferably at least 750 amino acids, orpreferably at least 775 amino acids. In another embodiment, preferredflea synaptic vesicle proteins comprise full-length proteins, i.e.,proteins encoded by full-length coding regions, or post-translationallymodified proteins thereof, such as mature proteins from which initiatingmethionine and/or signal sequences or “pro” sequences have been removed.

Additional preferred flea synaptic vesicle proteins of the presentinvention include proteins encoded by nucleic acid molecules comprisingat least a portion of nCfSVP1₁₈₇₅, nCfSVP1₁₅₉₀, nCfSVP2₃₃₁₄, and/ornCfSVP2₂₃₁₉, as well as flea synaptic vesicle proteins encoded byallelic variants of such nucleic acid molecules. A portion of such fleasynaptic vesicle nucleic acid molecule is preferably at least 30nucleotides in length.

Also preferred are flea synaptic vesicle proteins encoded by nucleicacid molecules having nucleic acid sequences comprising at least aportion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:8, and/or SEQ ID NO:11, as well as allelic variants of these nucleicacid molecules. A portion of such flea synaptic vesicle nucleic acidmolecule is preferably at least 30nucleotides in length.

In another embodiment, a preferred flea synaptic vesicle protein of thepresent invention is encoded by a nucleic acid molecule comprising atleast 20 nucleotides, preferably at least 25 nucleotides, preferably atleast 30 nucleotides, preferably at least 40 nucleotides, preferably atleast 50 nucleotides, preferably at least 75 nucleotides, preferably atleast 100 nucleotides, preferably at least 200 nucleotides, preferablyat least 400 nucleotides, preferably at least 500 nucleotides,preferably at least 750 nucleotides, preferably at least 1000nucleotides, preferably at least 1500 nucleotides, preferably at least1800 nucleotides, preferably at least 2000 nucleotides, preferably atleast 2300 nucleotides, or preferably at least 3300 nucleotides inlength. Within this embodiment is a flea synaptic vesicle proteinencoded by at least a portion of nCfSVP1₁₈₇₅, nCfSVP1₁₅₉₀, nCfSVP2₃₃₁₄,and/or nCfSVP2₂₃₁₉, or by an allelic variant of any of these nucleicacid molecules. Preferred flea synaptic vesicle proteins of the presentinvention are encoded by nucleic acid molecules comprising apparentlyfull-length flea synaptic vesicle coding region, i.e., nucleic acidmolecules encoding an apparently full-length flea synaptic vesicleprotein.

Preferred flea synaptic vesicle proteins of the present invention can beused to develop inhibitors that, when administered to an animal in aneffective manner, are capable of protecting that animal from fleainfestation. In accordance with the present invention, the ability of aninhibitor of the present invention to protect an animal from fleainfestation refers to the ability of that protein to, for example,treat, ameliorate and/or prevent infestation caused by fleas. Inparticular, the phrase “to protect an animal from flea infestation”refers to reducing the potential for flea population expansion on andaround the animal (i.e., reducing the flea burden). Preferably, the fleapopulation size is decreased, optimally to an extent that the animal isno longer bothered by fleas. A host animal, as used herein, is an animalfrom which fleas can feed by attaching to and feeding through the skinof the animal. Fleas, and other ectoparasites, can live on a host animalfor an extended period of time or can attach temporarily to an animal inorder to feed. At any given time, a certain percentage of a fleapopulation can be on a host animal whereas the remainder can be in theenvironment of the animal. Such an environment can include not onlyadult fleas, but also flea eggs and/or flea larvae. The environment canbe of any size such that fleas in the environment are able to jump ontoand off of a host animal. For example, the environment of an animal caninclude plants, such as crops, from which fleas infest an animal. Assuch, it is desirable not only to reduce the flea burden on an animalper se, but also to reduce the flea burden in the environment of theanimal.

Suitable fleas to target include any flea that is essentially incapableof causing disease in ail animal administered an inhibitor of thepresent invention. As such, fleas to target include any flea thatproduces a protein that can be targeted by an inhibitory compound thatinhibits a flea flea synaptic vesicle protein function, therebyresulting in the decreased ability of the parasite to cause disease inan animal. Preferred fleas to target include fleas of the followinggenera: Ctenocephalides, Cyopsyllus, Diamanus (Oropsylla), Echidnophaga,Nosopsyllus, Pulex, Tunga, and Xenopsylla, with those of the speciesCtenocephalides canis, Ctenocephalides felis, Diamanus montanus,Echidnophaga gallinacea, Nosopsyllus faciatus, Pulex irritans, Pulexsimulans, Tunga penetrans and Xenopsylla cheopis being more preferred,with C. felis being even more preferred. Such fleas are also preferredfor the isolation of proteins or nucleic acid molecules of the presentinvention.

One embodiment of a flea synaptic vesicle protein of the presentinvention is a fusion protein that includes a flea synaptic vesicleprotein-containing domain attached to one or more fusion segments.Suitable fusion segments for use with the present invention include, butare not limited to, segments that can: enhance a proteins stability; actas an immunopotentiator; and/or assist in purification of a fleasynaptic vesicle protein (e.g., by affinity chromatography). A suitablefusion segment can be a domain of any size that has the desired function(e.g., imparts increased stability, imparts increased immunogenicity toa protein, and/or simplifies purification of a protein). Fusion segmentscan be joined to amino and/or carboxyl termini of the flea synapticvesicle-containing domain of the protein and can be susceptible tocleavage in order to enable straight-forward recovery of a flea synapticvesicle protein. Fusion proteins are preferably produced by culturing arecombinant cell transformed with a fusion nucleic acid molecule thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of a flea synaptic vesicle-containingdomain. Preferred fusion segments include a metal binding domain (e.g.,a poly-histidine segment); an immunoglobulin binding domain (e.g.,Protein A; Protein G; T cell; B cell: Fc receptor or complement proteinantibody-binding domains): a sugar binding domain (e.g., a maltosebinding domain); and/or a “tag” domain (e.g., at least a portion ofβ-galactosidase, a strep tag peptide, a T7 tag peptide, a Flag™ peptide,or other domains that can be purified using compounds that bind to thedomain, such as monoclonal antibodies). More preferred fusion segmentsinclude metal binding domains, such as a poly-histidine segment; amaltose binding domain; a strep tag peptide, such as that available fromBiometra in Tampa, Fla.; and an S10 peptide.

The present invention also includes mimetopes of flea synaptic vesicleproteins of the present invention. As used herein, a mimetope of a fleasynaptic vesicle protein of the present invention refers to allycompound that is able to mimic the activity of such a flea synapticvesicle protein, often because the mimetope has a structure that mimicsthe particular flea synaptic vesicle protein. Mimetopes can be, but arenot limited to: peptides that have been modified to decrease thesusceptibility to degradation such as all-D retro peptides;anti-idiotypic and/or catalytic antibodies, or fragments thereof;non-proteinaceous immunogenic portions of an isolated protein (e.g.,carbohydrate structures); and synthetic or natural organic molecules,including nucleic acids. Such mimetopes can be designed usingcomputer-generated structures of proteins of the present invention.Mimetopes can also be obtained by generating random samples ofmolecules, such as oligonucleotides, peptides or other organicmolecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner.

Another embodiment of the present invention is an isolated nucleic acidmolecule comprising a flea synaptic vesicle nucleic acid molecule, i.e.a nucleic acid molecule that can be isolated from a flea cDNA library.As used herein, flea synaptic vesicle nucleic acid molecules has thesame meaning as flea synaptic vesicle nucleic acid molecule. Theidentifying characteristics of such nucleic acid molecules areheretofore described. A nucleic acid molecule of the present inventioncan include an isolated natural flea synaptic vesicle gene or ahomologue thereof, the latter of which is described in more detailbelow. A nucleic acid molecule of tile present invention can include oneor more regulatory regions, full-length or partial coding regions, orcombinations thereof. The minimal size of a nucleic acid molecule of thepresent invention is a size sufficient to allow the formation of astable hybrid (i.e., hybridization under stringent hybridizationconditions) with the complementary sequence of another nucleic acidmolecule. As such, the minimal size of a flea synaptic vesicle nucleicacid molecule of the present invention is from about 12 to about 18nucleotides in length.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subjected to human manipulation) andcan include DNA, RNA, or derivatives of either-DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. Isolated flea synaptic vesicle nucleic acidmolecules of the present invention, or homologues thereof, can beisolated from a natural source or produced using recombinant DNAtechnology (e.g., polymerase chain reaction (PCR) amplification orcloning) or chemical synthesis. Isolated flea synaptic vesicle nucleicacid molecules, and homologues thereof, can include, for example,natural allelic variants and nucleic acid molecules modified bynucleotide insertions, deletions, substitutions, and/or inversions in amanner such that the modifications do not substantially interfere withthe nucleic acid molecule's ability to encode a flea synaptic vesicleprotein of the present invention.

A flea synaptic vesicle nucleic acid molecule homologue can be producedusing a number of methods known to those skilled in the art, see, forexample, Sambrook et al., ibid. For example, nucleic acid molecules canbe modified using a variety of techniques including, but not limited to,classic mutagenesis and recombinant DNA techniques such as site-directedmutagenesis, chemical treatment, restriction enzyme cleavage, ligationof nucleic acid fragments, PCR amplification, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules, and combinations thereof. Nucleicacid molecule homologues can be selected by hybridization with fleasynaptic vesicle nucleic acid molecules or by screening the function ofa protein encoded by the nucleic acid molecule (e.g., ability to elicitan immune response against at least one epitope of a flea synapticvesicle protein or to effect flea synaptic vesicle activity).

An isolated flea synaptic vesicle nucleic acid molecule of the presentinvention call include a nucleic acid sequence that encodes at least oneflea synaptic vesicle protein of the present invention respectively,examples of such proteins being disclosed herein. Although the phrase“nucleic acid molecule” primarily refers to tile physical nucleic acidmolecule and the phrase “nucleic acid sequence” primarily refers to thesequence of nucleotides on the nucleic acid molecule, the two phrasescall be used interchangeably, especially with respect to a nucleic acidmolecule, or a nucleic acid sequence, being capable of encoding a fleasynaptic vesicle protein.

A preferred nucleic acid molecule or the present invention, whenadministered to an animal, is capable of protecting that animal fromflea infestation. As will be disclosed in more detail below, a nucleicacid molecule of the present invention can be, or encode, an antisenseRNA, a molecule capable of triple helix formation, a ribozyme, or othernucleic acid-based drug compound. In additional embodiments, a nucleicacid molecule of the present invention can encode a protective protein(e.g., a flea synaptic vesicle protein of the present invention), thenucleic acid molecule being delivered to the animal, for example, bydirect injection (i.e, as a genetic vaccine) or in a vehicle such as arecombinant virus vaccine or a recombinant cell vaccine.

In one embodiment of the present invention, a preferred flea synapticvesicle nucleic acid molecule includes an isolated nucleic acid moleculethat hybridizes under conditions that preferably allow less than orequal to 30% base pair mismatch, preferably under conditions that allowless than or equal to 20% base pair mismatch, preferably underconditions that allow less than or equal to 10% base pair mismatchpreferably under conditions that allow less than or equal to 5% basepair mismatch or preferably under conditions that allow less than orequal to 2% base pair mismatch with a nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:11, and/or SEQ ID NO:12.

Another embodiment of the present invention includes a flea synapticvesicle nucleic acid molecule, wherein said nucleic acid moleculehybridizes under conditions comprising, (a) hybridizing in solutioncomprising 1×SSC in the absence of nucleic acid helix destabilizingcompounds, at a temperature of 37° C. and (b) washing in a solutioncomprising 1×SSC in the absence of nucleic acid helix destabilizingcompounds, at a temperature of 47° C., to an isolated nucleic acidmolecule selected from the group consisting of S SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5. SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:11, and/or SEQ ID NO:12. Additional preferrednucleic acid molecules of the present invention include oligonucleotidesof an isolated nucleic acid molecule, wherein said nucleic acid moleculehybridizes under conditions comprising, (a) hybridizing in solutioncomprising 1×SSC in the absence of nucleic acid helix destabilizingcompounds, it a temperature of 37° C. and (b) washing in a solutioncomprising 1×SSC in the absence of nucleic acid helix destabilizingcompounds, at a temperature of 47° C., to an isolated nucleic acidmolecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7. SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:11, and/or SEQ ID NO:12, wherein said oligonucleotidecomprises at least 30nucleotides.

Additional preferred flea synaptic vesicle nucleic acid molecules of thepresent invention include nucleic acid molecules comprising a nucleicacid sequence that is preferably at least 70%, preferably at least 80%,preferably at least 90%, preferably at least 92%, preferably at least95%, or preferably at least 98% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:11, and/or SEQ ID NO:12. Also preferred are oligonucleotidesof any of such nucleic acid molecules. Percent identity as used hereinis determined using the Needleman-Wunsch algorithm, available in theSeqLab software using default parameters.

One embodiment of the present invention is a nucleic acid moleculecomprising all or part of nucleic acid molecules nCfSVP1₁₈₇₅,nCfSVP1₁₅₉₀, nCfSVP2₃₃₁₄, and/or nCfSVP2₂₃₁₉, or allelic variants ofthese nucleic acid molecules Another preferred nucleic acid molecule ofthe present invention includes at least a portion of nucleic acidsequence SEQ ID NO:1, SEQ ED NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and/or SEQID NO:12, as well as allelic variants of nucleic acid molecules havingthese nucleic acid sequences and homologues of nucleic acid moleculeshaving these nucleic acid sequences: preferably such a homologuesencodes or is complementary to a nucleic acid molecule that encodes atleast one epitope that elicits an immune response against a proteinhaving an amino acid sequence SEQ ID NO:3 and/or SEQ ID NO:9. Suchnucleic acid molecules acid include nucleotides in addition to thoseincluded in the SEQ ID NOs, such as, but not limited to, a full-lengthgene, a full-length coding region, a nucleic acid molecule encoding afusion protein, or a nucleic acid molecule encoding, a multivalentprotective compound.

In one embodiment, a flea synaptic vesicle nucleic acid molecule of thepresent invention encodes a protein having an amino acid sequence thatis at least 70%, preferably at least 80%, preferably at least 90%,preferably at least 95%, preferably at least 98%, preferably at least99%, or preferably at least 100% identical to SEQ ID NO:3 and/or SEQ IDNO:9. The present invention also includes a flea synaptic vesiclenucleic acid molecule encoding a protein having at least a portion ofSEQ ID NO:3 and/or SEQ ID NO:9, as well as allelic variants of a nucleicacid molecule encoding a protein having these sequences, includingnucleic acid molecules that have been modified to accommodate codonusage properties of the cells in which such nucleic acid molecules areto be expressed.

In another embodiment, a preferred flea synaptic vesicle nucleic acidmolecule of the present invention comprises a nucleic acid moleculecomprising at least 20 nucleotides, preferably at least 25 nucleotides,preferably at least 30 nucleotides, preferably at least 40 nucleotides,preferably at least 50 nucleotides, preferably at least 75 nucleotides,preferably at least 100 nucleotides, preferably at least 200nucleotides, preferably at least 400 nucleotides, preferably at least500 nucleotides, preferably at least 750 nucleotides, preferably atleast 1000 nucleotides, preferably at least 1500 nucleotides, preferablyat least 1800 nucleotides, preferably at least 2000 nucleotides,preferably at least 2300 nucleotides, or preferably at least 3300nucleotides in length.

In another embodiment, a preferred flea synaptic vesicle nucleic acidmolecule encodes a protein comprising at least 6 amino acids, preferablyat least 10 amino acids, preferably at least 20 amino acids, preferablyat least 30 amino acids, preferably at least 40 amino acids, preferablyat least 50 amino acids, preferably at least 75 amino acids, preferablyat least 100 amino acids, preferably at least 125 amino acids,preferably at least 150 amino acids, preferably at least 175 aminoacids, preferably at least 200 amino acids, preferably at least 250amino acids, preferably at least 300 amino acids, preferably at least350 amino acids, preferably at least 400 amino acids, preferably atleast 450 amino acids, preferably at least 500 amino acids, preferablyat least 550 amino acids, preferably at least 600 amino acids,preferably at least 650 amino acids, preferably at least 700 aminoacids, preferably at least 750 amino acids, or preferably at least 775amino acids.

In another embodiment, a preferred flea synaptic vesicle nucleic acidmolecule of the present invention comprises in apparently full-lengthflea synaptic vesicle coding region, i.e., the preferred nucleic acidmolecule encodes an apparently full-length flea synaptic vesicleprotein, respectively, or a post-translationally modified proteinthereof. In one embodiment, a preferred flea synaptic vesicle nucleicacid molecule of the present invention encodes a mature protein.

In another embodiment, a preferred flea synaptic vesicle nucleic acidmolecule of the present invention comprises a nucleic acid moleculecomprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and/or SEQID NO:12, or a fragment thereof.

A fragment of a flea synaptic vesicle nucleic acid molecule of thepresent invention preferably comprises at least 18 nucleotides,preferably at least 21 nucleotides, preferably at least 25 nucleotides,preferably at least 30 nucleotides, preferably at least 35 nucleotides,preferably at least 40 nucleotides, preferably at least 50 nucleotides,preferably at least 75 nucleotides, preferably at least 100 nucleotides,preferably at least 200 nucleotides, preferably at least 400nucleotides, preferably at least 500 nucleotides, preferably at least750 nucleotides, preferably at least 1000 nucleotides, preferably atleast 1500 nucleotides, preferably at least 1800 nucleotides, preferablyat least 2000 nucleotides, preferably at least 2300 nucleotides, orpreferably at least 3300 nucleotides identical in sequence to acorresponding contiguous sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and/or SEQ ID NO:12.

The phrase, a nucleic acid molecule comprising at least “x” contiguous,or consecutive nucleotides identical in sequence to at least “x”contiguous, or consecutive nucleotides of a nucleic acid moleculeselected from tile group consisting of SEQ ID NO:“y”, refers to an“x”-nucleotide in length nucleic acid molecule that is identical insequence to an “x”-nucleotide portion of SEQ ID NO:“y”, as well as tonucleic acid molecules that are longer in length than “x”. Theadditional length may be in the form of nucleotides that extend fromeither, tile 5′ or the 3′ end(s) of the contiguous identical“x”-nucleotide portion. The 5′ and/or 3′ extensions can include one ormore extensions that have no identity to a molecule of tile presentinvention, as well as extensions that show similarity or identity tocited nucleic acids sequences or portions thereof.

Knowing the nucleic acid sequences of certain flea synaptic vesiclenucleic acid molecules of the present invention allows one skilled inthe art to, for example, (a) make copies of those nucleic acidmolecules, (b) obtain nucleic acid molecules including at least aportion of such nucleic acid molecules (e.g., nucleic acid moleculesincluding full-length genes, full-length coding regions, regulatorycontrol sequences, truncated coding regions), and (c) obtain other fleasynaptic vesicle nucleic acid molecules. Such nucleic acid molecules canbe obtained in a variety of ways including screening appropriateexpression libraries with antibodies of the present invention;traditional cloning techniques using oligonucleotide probes of thepresent invention to screen appropriate libraries; and PCR amplificationof appropriate libraries or DNA using oligonucleotide primers of thepresent invention. Preferred libraries to screen or from which toamplify nucleic acid molecules include cDNA libraries as well as genomicDNA libraries. Similarly, preferred DNA sources to screen or from whichto amplify nucleic acid molecules include cDNA and genomic DNA.Techniques to clone and amplify genes are disclosed, for example, inSambrook et al., ibid.

The present invention also includes nucleic acid molecules that arcoligonucleotides capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingC. felis synaptic vesicle nucleic acid molecules or other flea synapticvesicle nucleic acid molecules. Oligonucleotides of the presentinvention can be RNA, DNA, or derivatives of either. The minimum size ofsuch oligonucleotides is the size required for formation of a stablehybrid between an oligonucleotide and a complementary sequence on anucleic acid molecule of tile present invention. A preferredoligonucleotide of the present invention has at maximum size ofpreferably 100 to 200 nucleotides. The present invention includesoligonucleotides that can be used as, for example, probes to identifynucleic acid molecules, primers to produce nucleic acid molecules, ortherapeutic reagents to inhibit flea synaptic vesicle protein productionor activity (e.g., as antisense-, triplex formation-, ribozyme- and/orRNA drug-based reagents). The present invention also includes tile useof such oligonucleotides to protect animals from disease using one ormore of such technologies. Appropriate oligonucleotide-containingtherapeutic compositions can be administered to an animal usingtechniques known to those skilled in the art.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulating of fleasynaptic vesicle nucleic acid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the molecule is able to be expressed whentransformed into a host cell. As used herein, an expression vector is aDNA or RNA vector that is capable of transforming a host cell and ofeffecting expression of a specified nucleic acid molecule. Preferably,the expression vector is also capable of replicating within the hostcell. Expression vectors can be either prokaryotic or eukaryotic, andare typically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, fungal, parasite, insect, other animal, and plant cells.Preferred expression vectors of the present invention call direct geneexpression in bacterial, yeast, insect and mammalian cells, and morepreferably in the cell types disclosed herein.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of tile presentinvention. In particular, recombinant molecules of tile presentinvention include transcription control sequences. Transcription controlsequences are sequences that control the initiation, elongation, andtermination of transcription. Particularly important transcriptioncontrol sequences ale those which control transcription initiation, suchas promoter, enhancer, operator and repressor sequences. Suitabletranscription control sequences include any transcription controlsequence that can function in at least one of the recombinant cells ofthe present invention. A variety of such transcription control sequencesare known to those skilled in the art. Preferred transcription controlsequences include those that function in bacterial, yeast, or insect andmammalian cells, such as, but not limited to, tac, lac, trp, trc,oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) andlambda p_(R) and fusions that include such promoters), bacteriophage T7,T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoter, antibiotic resistance gene, baculovirus, Heliothiszea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, otherpoxvirus, adenovirus, cytomegalovirus (such as immediate earlypromoter), simian virus 40, retrovirus, actin, retroviral long terminalrepeat, Rous sarcoma virus, heat shock, phosphate and nitratetranscription control sequences as well as other sequences capable ofcontrolling gene expression in prokaryotic or eukaryotic cells.Additional suitable transcription control sequences includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).Transcription control sequences of the present invention can alsoinclude naturally occurring transcription control sequences naturallyassociated with fleas, such as C. felis transcription control sequences.

Suitable and preferred nucleic acid molecules to include in recombinantvectors of the present invention are as disclosed herein. Preferrednucleic acid molecules to include in recombinant vectors, andparticularly in recombinant molecules, include nCfSVP1₁₈₇₅, nCfSVP1₁₅₉₀,nCfSVP2₃₃₁₄, and/or nCfSVP2₂₃₁₉.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable all expressed flea synaptic vesicle protein of the presentinvention to be secreted from the cell that produces the protein and/or(b) contain fusion sequences which lead to the expression of nucleicacid molecules of the present invention as fusion proteins. Examples ofsuitable signal segments include any signal segment capable of directingthe secretion of a protein of the present invention. Preferred signalsegments include, but are not limited to, tissue plasminogen activator(t-PA), interferon, interleukin, growth hormone, histocompatibility andviral envelope glycoprotein signal segments. Suitable fusion segmentsencoded by fusion segment nucleic acids are disclosed herein. Inaddition, a nucleic acid molecule of the present invention can be joinedto a fusion segment that directs the encoded protein to the proteosome,such as a ubiquitin fusion segment. Eukaryotic recombinant molecules mayalso include intervening and/or untranslated sequences surroundingand/or within the nucleic acid sequences of nucleic acid molecules ofthe present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. It is to be noted that a cellline refers to any recombinant cell of the present invention that is nota transgenic animal. Transformed nucleic acid molecules of the presentinvention can remain extrachromosomal or can integrate into one or moresites within a chromosome of the transformed (i.e., recombinant) cell insuch a manner that their ability to be expressed is retained. Preferrednucleic acid molecules with which to transform a cell include fleasynaptic vesicle nucleic acid molecules disclosed herein. Preferrednucleic acid molecules with which to transform a cell includenCfSVP1₁₈₇₅, nCfSVP1₁₅₉₀, nCfSVP2₃₃₁₄, and/or nCfSVP2₂₃₁₉.

Suitable host cells to transform include ally cell that can betransformed with it nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing flea synaptic vesicle proteins of thepresent invention or can be capable of producing such proteins afterbeing transformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal (including yeast), parasite (including helminth,protozoa and ectoparasite), other insect, other animal and plant cells.Preferred host cells include bacterial, mycobacterial, yeast, insect andmammalian cells. More preferred host cells include Salmonella,Escherichia, Bacillus, Caulobacter, Listeria, Saccharomyces, Pichia,Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells,MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandellfeline kidney cell line), CV-1 cells (African monkey kidney cell lineused, for example, to culture raccoon poxvirus), COS (e.g., COS-7)cells, and Vero cells. Particularly preferred host cells are Escherichiacoli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium, including attenuated strains such as UK-1 _(x)3987 andSR-11, _(x)4072; Caulobacter; Pichia; Spodoptera frugiperda;Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COScells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g.,ATCC CRL 1246). Additional appropriate mammalian cell hosts includeother kidney cell lines, other fibroblast cell lines (e.g., human,murine or chicken embryo fibroblast cell lines), myeloma cell lines,Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK³¹ cells and/orHeLa cells. In one embodiment, the proteins may be expressed asheterologous proteins in myeloma cell lines employing immunoglobulinpromoters.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences, examples of which are disclosed herein. The phraseoperatively linked refers to insertion of a nucleic acid molecule intoan expression vector in a manner such that the molecule is able to beexpressed when transformed into a host cell.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transfercells are disclosed herein.

Recombinant cells of the present invention can also be co-transformedwith one or more recombinant molecules including flea synaptic vesiclenucleic acid molecules encoding one or more proteins of the presentinvention and one or more other nucleic acid molecules encoding otherprotective compounds, as disclosed herein (e.g., to produce multivalentvaccines).

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes., additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, deletion ofsequences that destabilize transcripts, and use of control signals thattemporally separate recombinant cell growth from recombinant enzymeproduction during fermentation. The activity of all expressedrecombinant protein of the present invention may be improved byfragmenting, modifying, or derivatizing nucleic acid molecules encodingsuch a protein.

Isolated flea synaptic vesicle proteins of the present invention can beproduced in a variety of ways, including production and recovery ofnatural proteins, production and recovery of recombinant proteins, andchemical synthesis of the proteins. In one embodiment, an isolatedprotein of the present invention is produced by culturing a cell capableof expressing the protein under conditions effective to produce theprotein, and recovering the protein. A preferred cell to culture is arecombinant cell of the present invention. Effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Aneffective, medium refers to any medium in which a cell is cultured toproduce a flea synaptic vesicle protein of the present invention. Suchmedium typically comprises an aqueous medium having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. Cells of the present inventioncan be cultured in conventional fermentation bioreactors, shake flasks,test tubes, microtiter dishes, and petri plates. Culturing can becarried out at a temperature, pH and oxygen content appropriate for arecombinant cell. Such culturing conditions are within the expertise ofone of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane.

The phrase “recovering the protein”, as well as similar phrases, refersto collecting the whole fermentation medium containing the protein andneed not imply additional steps of separation or purification. Proteinsof the present invention can be purified using a variety of standardprotein purification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization,Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of stimulating the production of antibodiesin a treated animal.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to a flea synapticvesicle protein of the present invention or a mimetope thereof (e.g.,anti-flea synaptic vesicle antibodies). As used herein, the term“selectively binds to” a protein refers to the ability of antibodies oftile present invention to preferentially bind to specified proteins andmimetopes thereof of the present invention. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example,Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, aLaboratory. Manual, Cold Spring Harbor Labs Press; Harlow et al. ibid.An anti-flea synaptic vesicle antibody of the present inventionpreferably selectively binds to a flea synaptic vesicle protein,respectively, in such a way as to inhibit the function of that protein.

Isolated antibodies of the present invention can include antibodies inserum, o antibodies that have been purified to varying degrees.Antibodies of the present invention can be polyclonal or monoclonal, orcan be functional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies orchimeric antibodies that can bind to one or more epitopes.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce flea synaptic vesicleproteins of the present invention. Antibodies raised against definedproteins or mimetopes can be advantageous because such antibodies arenot substantially contaminated with antibodies against other substancesthat might otherwise cause interference in a diagnostic assay or sideeffects if used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as therapeutic compounds to passivelyimmunize an animal in order to protect the animal from fleas susceptibleto treatment by such antibodies and/or (b) as tools to screen expressionlibraries and/or to recover desired proteins of tile present inventionfrom a mixture of proteins and other contaminants. Furthermore,antibodies of the present invention can be used to target cytotoxicagents to fleas in order to directly kill such fleas. Targeting can beaccomplished by conjugating (i.e., stably joining) such antibodies tothe cytotoxic agents using techniques known to those skilled in the art.Suitable cytotoxic agents are known to those skilled in the art.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal susceptible to flea infestation, iscapable of protecting that animal from flea infestation. Therapeuticcompositions of the present invention include at least one of thefollowing protective molecules: an isolated flea synaptic vesicleprotein; a mimetope of an isolated flea synaptic vesicle protein; anisolated flea synaptic vesicle nucleic acid molecule; and/or a compoundderived from said isolated flea synaptic vesicle protein that inhibitsflea synaptic vesicle protein activity. A therapeutic composition of thepresent invention can further comprise a component selected from thegroup of an excipient, a carrier, and/or an adjuvant; these componentsare described further herein. As used herein, a protective molecule orprotective compound refers to a compound that, when administered to ananimal in an effective manner, is able to treat, ameliorate, and/orprevent flea infestation. Preferred fleas to target are heretoforedisclosed. One example of a protective molecule is a vaccine, such as,but not limited to, a naked nucleic acid vaccine, a recombinant virusvaccine, a recombinant cell vaccine, and a recombinant protein vaccine.Another example of a protective molecule is a compound that inhibitsflea synaptic vesicle protein activity, such as an isolated antibodythat selectively binds to a flea synaptic vesicle protein, a substrateanalog of a flea synaptic vesicle protein, anti-sense-, triplexformation-, ribozyme-, and/or RNA drug-based compounds, or otherinorganic or organic molecules that inhibit flea synaptic vesicleprotein activity. Inhibiting flea synaptic vesicle protein activity canrefer to the ability of a compound to reduce the activity of fleasynaptic vesicle proteins. Inhibiting flea synaptic vesicle proteinactivity can also refer to the ability of a compound to reduce theamount of flea synaptic vesicle protein in a flea.

One embodiment of the present invention is a therapeutic compositioncomprising an excipient and a compound selected from tile groupconsisting of: (a) an isolated nucleic acid molecule selected from thegroup consisting of a flea cDNA molecule and a flea mRNA molecule,wherein said nucleic acid molecule is at least 30 nucleotides in lengthand hybridizes with a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:11, and SEQ ID NO:12, under conditions comprising (I)hybridizing in a solution comprising 1×SSC in the absence of nucleicacid helix destabilizing compounds, at a temperature of 37° C. and (2)washing in a solution comprising 1×SSC in the absence of helixdestabilizing compounds, at a temperature of 47° C.; (b) an isolatedprotein encoded by a nucleic acid molecule at least 30 nucleotides inlength that hybridizes with a nucleic acid molecule having a nucleicacid sequence selected from the group consisting of SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:10, and/or SEQ ID NO:12, under conditions comprising (i)hybridizing in a solution comprising 1×SSC in the absence of helixdestabilizing compounds, at a temperature of 37° C. and (ii) washing ina solution comprising 1×SSC in the absence of helix destabilizingcompounds, at a temperature of 47° C.; and (c) an isolated antibody thatselectively binds to a protein of (b).

Another embodiment of the present invention includes a method to reduceflea infestation in an animal susceptible to flea infestation. Such amethod includes the step of administering to the animal a therapeuticmolecule comprising a protective compound selected from the groupconsisting, of (a) an isolated flea synaptic vesicle protein; (b) amimetope of an isolated flea synaptic vesicle protein; (c) an isolatedflea synaptic vesicle nucleic acid molecule; and (d) a compound derivedfrom an isolated flea synaptic vesicle protein that inhibits fleasynaptic vesicle protein activity.

Therapeutic compositions of the present invention can be administered toany animal susceptible to flea infestation, preferably to mammals, andmore preferably to dogs, cats, humans, ferrets, horses, cattle, sheep,and other pets, economic food animals, work animals and/or zoo animals.Preferred animals to protect against flea infestation include dogs,cats, humans, and ferrets, with dogs and cats being particularlypreferred.

As used herein, the term derived, or the ter-m derived from, refers to apeptide, antibody, mimetope, nucleic acid molecule, or other compoundthat was obtained from or obtained using a flea synaptic vesicle proteinor nucleic acid molecule of the present invention. Methods to obtainderivatives from a flea synaptic vesicle molecule of the presentinvention are known in the art, and as such include, but are not limitedto molecular modeling of flea synaptic vesicle proteins to determineactive sites, and predicting from these active sites smaller fragmentsand/or mimetopes that retain and/or mimic these active sites, therebyinhibiting flea synaptic vesicle protein activity. Other inhibitors offlea synaptic vesicle activity can also be obtained in a variety ofways, including but not limited to screening of peptide or smallchemical compound libraries against flea synaptic vesicle proteins ofthe present invention; and screening of polyclonal or monoclonalantibodies to find antibodies that specifically bind flea synapticvesicle proteins of the present invention.

A flea synaptic vesicle protein inhibitor of the present invention (i.e.an inhibitor of a flea synaptic vesicle protein) is identified by itsability to mimic, bind to, modify, or otherwise interact with, a fleasynaptic vesicle protein, thereby inhibiting the activity of a naturalflea synaptic vesicle protein. Suitable inhibitors of flea synapticvesicle protein activity are compounds that inhibit flea synapticvesicle protein activity in at least one of a variety of ways: (a) bybinding to or otherwise interacting with or otherwise modifying fleasynaptic vesicle protein sites; (b) by binding to the flea synapticvesicle protein and thus reducing the availability of the flea synapticvesicle protein in solution; (c) by mimicking a flea synaptic vesicleprotein; and (d) by inter-acting with other regions of the flea synapticvesicle protein to inhibit flea synaptic vesicle protein activity, forexample, by allosteric interaction.

Flea synaptic vesicle protein inhibitor-s can be used directly ascompounds in compositions of the present invention to treat animals aslong as such compounds are not harmful to host animals being treated.Preferred flea synaptic vesicle protein inhibitors of the presentinvention include, but are not limited to, flea synaptic vesicle proteinsubstrate analogs, and other molecules that bind to a flea synapticvesicle protein (e.g., to an allosteric site) in such a manner that theactivity of the flea synaptic vesicle protein is inhibited. A fleasynaptic vesicle protein substrate analog refers to a compound thatinteracts with (e.g., binds to, associates with, modifies) the activesite of a flea synaptic vesicle protein. A preferred flea synapticvesicle protein substrate analog inhibits flea synaptic vesicle proteinactivity. Flea synaptic vesicle protein substrate analogs can be of anyinorganic or organic composition. Flea synaptic vesicle proteinsubstrate analogs can be, but need not be, structurally similar to aflea synaptic vesicle protein natural substrate as long as they caninteract with the active site of that flea synaptic vesicle protein.Flea synaptic vesicle protein substrate analogs can be designed usingcomputer-generated structures of flea synaptic vesicle proteins of thepresent invention or computer structures of flea synaptic vesicleprotein's natural substrates. Preferred sites to model include one ormore of the active sites of flea synaptic vesicle proteins. Substrateanalogs can also be obtained by generating random samples of molecules,such as oligonucleotides, peptides, peptidomimetic compounds, or otherinorganic or organic molecules, and screening such samples for theirability to interfere with interaction between flea synaptic vesicleproteins and their substrates, e.g. by affinity chromatographytechniques. A preferred flea synaptic vesicle protein substrate analogis a flea synaptic vesicle protein mimetic compound, i.e., a compoundthat is structurally and/or Functionally similar to a natural substrateof a flea synaptic vesicle protein of the present invention,particularly to the region of the substrate that interacts with the fleasynaptic vesicle protein active site, but that inhibits flea synapticvesicle protein activity upon interacting with the flea synaptic vesicleprotein active site.

The present invention also includes a therapeutic composition comprisingat least one protective molecule of the present invention in combinationwith at least one additional compound protective against one or moreinfectious agents.

In one embodiment, a therapeutic composition of the present inventioncan be used to protect an animal from flea infestation by administeringsuch composition to a flea in order to prevent infestation. Suchadministration to the flea and/or animal could be oral, or byapplication to the animal's body surface (e.g. topical spot-on, orspraying onto the animal), or by application to the environment (e.g.,spraying). Examples of such compositions include, but are not limitedto, transgenic vectors capable of producing at least onic therapeuticcomposition of the present invention. In another embodiment a flea caningest therapeutic compositions, or products thereof, present on thesurface of or in the blood of a host animal that has been administered atherapeutic composition of the present invention.

In accordance with the present invention, a host animal (i.e., an animalthat is or is capable of being infested with fleas) is treated byadministering to the animal a therapeutic composition of the presentinvention in such a manner that the composition itself (e.g., a fleasynaptic vesicle protein, a flea synaptic vesicle nucleic acid molecule,a flea synaptic vesicle protein inhibitor, a synaptic vesicle proteinsynthesis suppressor (i.e., a compound that decreases the production orhalf-life of a synaptic vesicle protein in fleas), a flea synapticvesicle protein mimetope, or a anti-flea synaptic vesicle antibody) or aproduct generated by the animal in response to administration of thecomposition (e.g., antibodies produced in response to administration ofa flea synaptic vesicle protein or nucleic acid molecule, or conversionof an inactive inhibitor “prodrug” to an active flea synaptic vesicleprotein inhibitor) ultimately enters the flea. A host animal ispreferably treated in such a way that the compound or product thereof ispresent on the body surface of the animal or enters the blood stream ofthe animal. Fleas are then exposed to the composition or product whenthey feed from the animal. For example, flea synaptic vesicle proteininhibitors administered to an animal are administered in such a way thatthe inhibitors enter the blood stream of the animal, where they can betaken up by feeding fleas.

The present invention also includes the ability to reduce larval fleainfestation in that when fleas feed from a host animal that has beenadministered a therapeutic composition of the present invention, atleast a portion of compounds of the present invention, or productsthereof in the blood taken up by the fleas are excreted by the fleas infeces, which is subsequently ingested by flea larvae. In particular, itis of note that flea larvae obtain most, if not all, of their nutritionfrom flea feces.

In accordance with the present invention, reducing flea synaptic vesicleprotein activity in a flea call lead to a number of outcomes that reduceflea burden on treated animals and their surrounding environments. Suchoutcomes include, but are not limited to, (a) reducing the viability offleas that feed from the treated animal, (b) reducing the fecundity offemale fleas that feed from the treated animal, (c) reducing thereproductive capacity of male fleas that feed from the treated animal,(d) reducing the viability of eggs laid by female fleas that feed fromthe treated animal, (e) altering the blood feeding behavior of fleasthat feed from the treated animal (e.g., fleas take up less volume perfeeding or feed less frequently), (f) reducing the viability of flealarvae, for example due to the feeding of larvae from feces of fleasthat feed from the treated animal, (g) altering the development of flealarvae (e.g., by decreasing feeding behavior, inhibiting growth,inhibiting (e.g., slowing or blocking) molting, and/or otherwiseinhibiting maturation to adults), and/or (h) altering or decreasing theability of fleas or flea larvae to digest a blood meal.

In order to protect an animal from flea infestation, a therapeuticcomposition of the present invention is administered to the animal in aneffective manner such that the composition is capable of protecting thatanimal from flea infestation. Therapeutic compositions of the presentinvention can be administered to animals prior to infestation in orderto prevent infestation (i.e., as a preventative vaccine) and/or can beadministered to animals after infestation. For example, proteins,mimetopes thereof, and antibodies thereof can be used asimmunotherapeutic agents.

Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextrin. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, or o-cresol, formalin and benzylalcohol. Standard formulations call either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, serum albumin, preservatives, etc., to whichsterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncall include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), Flt-3 ligand, granulocyte colony stimulating factor (G-CSF),macrophage colony stimulating factor (M-CSF), colony stimulating factor(CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (L-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interferon gamma, interferon gamma inducingfactor I (IGIF), transforming growth factor beta, RANTES (regulated uponactivation, normal T cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF)); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts, calcium-based salts; silica;polynucleotides; toxoids; serum proteins, viral coat proteins: blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of thc present invention is a controlled releaseformulation that is capable of slowly releasing, a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition. The therapeutic compositionis preferably released over a period of time ranging from 1 to 12months. A controlled release formulation of the present invention iscapable of effecting a treatment preferably for at least 1 month,preferably for at least 3 months, preferably for at least 6 months,preferably for at least 9 months, and preferably for at least 12 months.

Acceptable protocols to administer therapeutic compositions in aneffective manner include individual dose size, number of doses,frequency of dose administration, and mode of administration.Determination of such protocols can be accomplished by those skilled inthe art. A suitable single dose is a dose that is capable of treating ananimal when administered one or more times over a suitable time period.For example, a preferred single dose of an inhibitor is from about 1microgram (μg) to about 10 milligrams (mg) of the therapeuticcomposition per kilogram body weight of the animal. Booster vaccinationscan be administered from about 2 weeks to several years after theoriginal administration. Booster administrations preferably arcadministered when the immune response of the animal becomes insufficientto protect the animal from disease. A preferred administration scheduleis one in which from about 10 μg to about 1 mg of the therapeuticcomposition per kg body sleight of the animal is administered from aboutone to about two times over a time period of from about 2 weeks to about12 months. Modes of administration can include, but are not limited to,subcutaneous, intradermal, intravenous, intranasal, oral, transdermal,intraocular, intranasal, conjunctival, and intramuscular routes. Methodsof administration for other therapeutic compounds can be determined byone skilled in the art, and nay include administration of a therapeuticcomposition one or more times, on a daily, weekly, monthly or yearlyregimen; routes of administration can be determined by one skilled inthe art, and may include any route. A preferred route of administrationof an inhibitory compound when administering to fleas is a topical, or“spot-on” formulation administered to the body surface of the animal, sothat a flea would encounter the inhibitory compound when attached to theanimal; another preferred route of administration of an inhibitorycompound is an oral formulation that, when fed to an animal, would enterthe bloodstream of the animal, which would then be transferred to a fleawhile feeding from the animal.

A recombinant protein vaccine of the present invention comprises arecombinantly-produced flea synaptic vesicle protein of the presentinvention that is administered to an animal according to a protocol thatresults in the animal producing a sufficient immune response to protectitself from a flea infestation. Such protocols can be determined bythose skilled in the art.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein orprotective RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNAdrug) in the animal. Nucleic acid molecules can be delivered to ananimal in a variety of methods including, but not limited to, (a)administering a naked (i.e., not packaged in a viral coat or cellularmembrane) nucleic acid as a genetic vaccine (e.g., as naked DNA or RNAmolecules, such as is taught, for example in Wolff et al., 1990, Science247. 1465-1468) or (b) administering a nucleic acid molecule packaged asa recombinant virus vaccine or as a recombinant cell vaccine (i.e., thenucleic acid molecule is delivered by a viral or cellular vehicle).

A genetic (i.e., naked nucleic acid) vaccine of tie present inventionincludes a nucleic acid molecule of the present invention and preferablyincludes a recombinant molecule of the present invention that preferablyis replication, or otherwise amplifications competent. A genetic vaccineof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a dicistronicrecombinant molecule. Preferred genetic vaccines include at least aportion of a viral genome, i.e., a viral vector. Prefer-red viralvectors include those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses, and retroviruses, with those based onalphaviruses, such as sindbis or Semliki forest virus, species-specificherpesviruses and poxviruses being particularly preferred. Any suitabletranscription control sequence can be used, including those disclosed assuitable for protein production. Particularly preferred transcriptioncontrol sequences include cytomegalovirus immediate early (preferably inconjunction with Intron-A), Rous sarcoma virus long terminal repeat, andtissue-specific transcription control sequences, as well astranscription control sequences endogenous to viral vectors if viralvectors are used. The incorporation of a “strong” polyadenylation signalis also preferred.

Genetic vaccines of the present invention can be administered in avariety of ways, with intramuscular, subcutaneous, intradermal,transdermal, conjunctival, intraocular, intranasal and oral routes ofadministration being preferred. A preferred single dose of a geneticvaccine ranges from about 1 nanogram (ng) to about 600 μg, depending onthe route of administration and/or method of delivery, as can bedetermined by those skilled in the art. Suitable delivery methodsinclude, for example, by injection, as drops, aerosolized and/ortopically. Genetic vaccines of the present invention can be contained inan aqueous excipient (e.g., phosphate buffered saline) alone or in acarrier (e.g., lipid-based vehicles).

A recombinant virus vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging- orreplication-deficient and/or encodes (in attenuated virus. A number ofrecombinant viruses can be used, including, but not limited to, thosebased on alplhaviruses, poxviruses, adenoviruses, herpesviruses,picornaviruses, and retroviruses. Preferred recombinant virus vaccinesare those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, species-specific herpesviruses and species-specificpoxviruses. An example of methods to produce and use alphavirusrecombinant virus vaccines are disclosed in U.S. Pat. No. 5,766,602 toXiong, and Grieve.

When administered to an animal, a recombinant virus vaccine of thepresent invention infects cells within the immunized animal and directsthe production of a protective protein or RNA nucleic acid molecule thatis capable of protecting the animal from flea infestation as disclosedherein. For example, a recombinant virus vaccine comprising a fleasynaptic vesicle nucleic acid molecule of the present invention isadministered according to a protocol that results in the animalproducing a sufficient immune response to protect itself from fleainfestation. A preferred single dose of a recombinant virus vaccine ofthe present invention is from about 1×10⁴ to about 1×10⁸ virus plaqueforming units (pfu) per kilogram body weight of the animal.Administration protocols are similar to those described herein forprotein-based vaccines, with subcutaneous, intramuscular, intranasal,intraocular, conjunctival, and oral administration routes beingpreferred.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one protein of thepresent invention. Preferred recombinant cells for this embodimentinclude Salmonella, E, coli, Listeria, Mycobacterium, S. frugiperda,yeast, (including Saccharomyces cerevisiae and Pichia pastoris), BHK,CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinantcells. Recombinant cell vaccines of the present invention can beadministered in a variety of ways but have the advantage that they canbe administered orally, preferably at doses ranging from about 10⁸ toabout 10¹² cells per kilogram body weight. Administration protocols aresimilar to those described herein for protein-based vaccines.Recombinant cell vaccines can comprise whole cells, cells stripped ofcell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention toprotect an animal from flea infestation can be tested in a variety ofways including, but not limited to, detection of protective antibodies(using, for example, proteins or mimetopes of the present invention),detection of cellular immunity within tile treated animal, or challengeof the treated animal with the fleas to determine whether the treatedanimal is resistant to infestation. Challenge studies call includedirect administration of fleas to the treated animal. In one embodiment,therapeutic compositions call be tested in animal models such as mice.Such techniques are known to those skilled in the art.

As discussed herein, one therapeutic composition of the presentinvention includes an inhibitor of flea synaptic vesicle proteinactivity, i.e., a compound capable of substantially interfering with thefunction of a flea synaptic vesicle protein. An inhibitor of fleasynaptic vesicle protein activity, or function, can be identified usingflea synaptic vesicle proteins of the present invention. A preferredinhibitor of flea synaptic vesicle protein function is a compoundcapable of substantially interfering with the function of a fleasynaptic vesicle protein and which does not substantially interfere withthe function of host animal synaptic vesicle proteins. As used herein, acompound that does not substantially inhibit or interfere with hostanimal synaptic vesicle proteins is one that, when administered to ahost animal, tile host animal shows no significant adverse effectsattributable to the inhibition of synaptic vesicle and which, whenadministered to an animal in an effective manner, is capable ofprotecting that animal from flea infestation.

One embodiment of the present invention is a method to identify acompound capable of inhibiting flea synaptic vesicle protein activity.Such a method includes the steps of (a) contacting (e.g., combining,mixing) an isolated flea synaptic vesicle protein of the presentinvention, with a putative inhibitory compound under conditions inwhich, in the absence of the compound, the protein has flea synapticvesicle protein activity, and (b) determining if the putative inhibitorycompound inhibits the activity. Flea synaptic vesicle protein activitycan be determined in a variety of ways known in the art, including butnot limited to determining the ability of flea synaptic vesicle proteinto bind to or otherwise interact with a substrate. Such conditions underwhich a flea synaptic vesicle protein has flea synaptic vesicle proteinactivity include conditions in which a flea synaptic vesicle protein hasa correct three-dimensionally folded structure under physiologicconditions, i.e. physiologic pH, physiologic ionic concentrations, andphysiologic temperatures.

Putative inhibitory compounds to screen include antibodies (includingfragments and mimetopes thereof), putative substrate analogs, and other,preferably small, organic or inorganic molecules. Methods to determineflea synaptic vesicle protein activity are known to those skilled in theart.

A preferred method to identify a compound capable of inhibiting fleasynaptic vesicle protein activity includes contacting an isolated fleasynaptic vesicle protein of the present invention with a putativeinhibitory compound under conditions in which, in the absence of thecompound, the protein has flea synaptic vesicle protein activity; anddetermining if the putative inhibitory compound inhibits the activity.

Another embodiment of the present invention is an assay kit to identifyan inhibitor of a flea synaptic vesicle protein of the presentinvention. This kit comprises an isolated flea synaptic vesicle proteinof the present invention, and a means for determining inhibition of anactivity of flea synaptic vesicle protein, where the means enablesdetection of inhibition. Detection of inhibition of flea synapticvesicle protein identifies a putative inhibitor to be an inhibitor of aflea synaptic vesicle protein. Means for determining inhibition of aflea synaptic vesicle protein include, for example, an assay system thatdetects binding of a putative inhibitor to a flea synaptic vesiclemolecule, and an assay system that detects interference by a putativeinhibitor of the ability of flea synaptic vesicle protein to hydrolyze asubstrate. Means and methods are described herein and are known to thoseskilled in the art.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention. Thefollowing examples include a number of recombinant DNA and proteinchemistry techniques known to those skilled in the art; see, forexample, Sambrook et al., ibid.

Example 1

This Example describes the isolation of RNA from the hindgut andMalpighian tubules (HMT) of Ctenocephalides felis and the use ofisolated RNA to construct subtracted and unsubtracted cDNA libraries.

Approximately 10,000 hindguts and Malpighian tubules were dissected fromequal numbers of cat blood fed and unfed adult C. felis with a male tofemale ratio of 1 to 4, and total RNA was extracted using a guanidineisothiocyanate lysis buffer and the standard procedure described bySambrook et al. Poly-A enriched mRNA was purified from total RNA aboveusing a mRNA Purification Kit, available from Pharmacia Biotech,Piscataway, N. J., following, the manufacturer's protocol. The sameprocedures ere used to extract total RNA and isolate poly-A enrichedmRNA from the dissected C. felis bodies following removal of HMT,referred to hereinafter as “non-HMT mRNA”.

Poly-A enriched mRNA was used to construct a cDNA library usingsubtractive hybridization and suppression PCR as follows. Subtractivehybridization and suppression PCR was conducted using a PCR-Select™ cDNASubtraction Kit, available from Clontech Laboratories, Inc., Palo Alto,Calif. according to the manufacturer's instructions. Briefly, this kituses subtractive hybridization and suppression PCR to specificallyamplify cDNA sequences that are present in the tester cDNA and absent inthe driver cDNA, thus clinching for tester-specific sequences. Theefficiency of the subtraction process can be assessed bysemi-quantitative PCR and by comparing the ethidium bromide stainingpatterns of the subtracted and unsubtracted samples on agarose gels asdescribed in section V. D. of the manufacturer's protocol. For thesemi-quantitative PCR, three genes with mRNAs known to be expressedoutside of the HMT tissue were used to test for specific subtraction.These genes encoded putative actin, N-aminopeptidase, and serineprotease proteins.

Subtractive hybridization and suppression PCR was conducted under thefollowing conditions. Two micrograms (μg) of HMT mRNA was used as thetemplate for synthesis of the tester material and 2 μg of non-HMT mRNAwas used as template for synthesis of the driver material in thisreaction. The number of cycles used in the selective amplification stepswas optimized using the manufacturer's protocols. Optimization resultedin the use of 24 rather than the standard 27 cycles of primary PCR incombination with 15 cycles of secondary PCR rather than the standard 12cycles.

The products from the suppressive PCR reaction were ligated into thepCR®2.1 vector, available from Invitrogen, Carlsbad, Calif., using anOriginal TA Cloning® Kit, available from Invitrogen. The ligationreaction was then used to transform INVαF One Shot™ competent cells,available from Invitrogen, which were plated on Luria broth (LB) agarwith 50 micrograms per milliliter (μg/l) ampicillin, available fromSigma-Aldrich Co. St. Louis, Mo., and 50 μg/ml 5-bromo-4-chloro-3-indoylβ-D-galactopyranoside (X-Gal), available from Fisher Biotech, Fair Lawn,N.J. Transformed colonies were amplified and the DNA isolated using thestandard alkaline lysis procedure described by Sambrook et al., ibid.

Automated cycle sequencing of DNA samples was performed using all ABIPRISM™ Model 377, available from Perkins Elmer, with XL upgrade DNASequencer, available from PE Applied Biosystems, Foster City, Calif.,after reactions were carried out using the PRISM™ Dye Terminator CycleSequencing Ready Reaction Kit or the PRISM™ dRhodamine Terminator CycleSequencing Ready Reaction Kit or the PRISM™ BigDye™ Terminator Cyclesequencing Ready Reaction Kit, available from PE Applied Biosystems,following the manufacturer's protocol, hereinafter “standard sequencingmethods”. Sequence analysis was performed using SeqLab, using defaultparameters. Each sequence read was trimmed of vector sequence at eitherend and submitted for a search through the National Center forBiotechnology Information (NCBI), National Library of Medicine, NationalInstitute of Health, Baltimore, Md., using the BLAST network. Thisdatabase includes SwissProt+PIR+SPupdate+GenPept+GPUpdate+PDB databases.The search was conducted using the xBLAST function, which compares thetranslated sequences in all 6 reading frames to the protein sequencescontained in the database.

An unsubtracted HMT cDNA library was constructed as follows.Approximately 10,000 HMT tissues were dissected from equal numbers ofunfed and cat blood-fed adult C. felis with a male to female ratio of1:4. Total RNA was extracted using a guanidine isothiocyanate lysisbuffer and procedures described in Sambrook et al., followed byisolation using a mRNA purification kit, available from Pharmacia,according to the manufacturer's protocols. The library was constructedwith 5 μg of isolated mRNA using a ZAP-cDNA®cDNA synthesis kit, andpackaged using a ZAP-cDNA®Gigapack® gold cloning kit, both availablefrom Stratagene, La Jolla, Calif. The resultant HMT library wasamplified to a titer of about 5×10⁹ plaque forming units per milliliter(pfu/ml). Single clone excisions were performed using the Ex-Assist™helper phage, available from Stratagene, and used to create doublestranded plasmid template for sequencing using the manufacturer'sprotocols with the following exceptions. Following incubation of theSOLR cells with the cleared phage lysate, the mixture was used toinoculate LB broth, and the mix was incubated overnight and thensubjected to mini-prep plasmid preparation and sequencing as describedfor the subtracted HMT library above.

Example 2

This Example describes the further characterization of synaptic vesicle2B-like sequence nucleic acid molecules of the present invention.

A cDNA, designated clone 2104-59, was isolated from the subtracted HMTlibrary as described in Example 1; the nucleic acid sequence of thecDNA's coding strand is denoted herein as SEQ ID NO:1. DNA from clone2104-59 was purified, and the insert used for plaque hybridizationscreening of the unsubtracted HMT cDNA library as follows. The insertfrom clone 2104-59 was excised by digestion with EcoRI, separated byagarose gel electrophoresis and purified using the QiaQuick GelExtraction kit, available from Qiagen. A Megaprime DNA labeling kit,available from Amersham Pharmacia, was used to incorporate α-³²P-labeleddATP into the random-primed probe mix. The ³²P α-dATP labeled probe wasused in a plaque lift hybridization as follows. Filters were hybridizedwith about 1×10⁶ counts per minute (cpm) per ml of tile probe in 5×SSPE,(see Sambrook et al., ibid.), 1.2% sodium dodecyl sulfate (SDS), 0.1mg/ml salmon sperm DNA and 5× Denhardt's reagent, (see Sambrook et al.,ibid.), at 55° C. for about 14 hours. The filters were washed asfollows: (a) 10 minutes with 5×SSPE and 1% SDS, (b) 10 minutes with2×SSPE and 1% SDS, (c) 10 minutes with 1×SSPE and 0.5% SDS, and (d) 10minutes with 0.5×SSPE and 1% SDS. All washes were conducted at 55° C.Plaques that hybridized strongly to the probe were isolated andsubjected to in vivo excision. In vivo excision was performed using theStratagene Ex-Assist™ helper phage system and protocols, available fromStratagene, to convert a positive plaque to pBluescript™ plasmid DNA.Sequencing was conducted using standard sequencing methods followingpreparation of DNA with a Qiagen Qiaprep™ spin mini prep kit, availablefrom Qiagen, using the manufacturer's instructions and restrictionenzyme digestion with about 1 μl of 20 U/μl each of EcoRI and XhoI,available from New England Biolabs, Beverly, Mass.

Hybridization and plaque purification resulted in the isolation of aclone containing, an about 1875 nucleotide synaptic vesicle 2B-likesequence, referred to herein as nCfSVP1₁₈₇₅, with a coding stranddenoted nucleic acid sequence SEQ ID NO:2 and a complementary sequencedenoted SEQ ID NO:4. Translation of SEQ ID NO:2 suggests that nucleicacid molecule nCfSVP1₁₈₇₅ encodes a full-length synaptic vesicle 2B-likeprotein of 530 amino acids, referred to herein as PCfSVP1₅₃₀ with anamino acid sequence represented by SEQ ID NO:3, assuming the initiationcodon spans from nucleotide 44 through nucleotide 46 of SEQ ID NO:2 andthe termination codon spans from nucleotide 1634 through nucleotide 1636of SEQ ID NO:2. The coding region encoding PCfSVP1₅₃₀, is represented bynucleic acid molecule nCfSVP1₁₅₉₀, with a coding strand with the nucleicacid sequence denoted SEQ ID NO:5 and a complementary strand withnucleic acid sequence denoted SEQ ID NO:6. The amino acid sequence ofSEQ ID NO:3, predicts that PCfSVP1₅₃₀ has an estimated molecular weightof about 58.7 kDa and an isoelectric point (pI) of about 7.61.

Comparison of amino acid sequence SEQ ID NO:3 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:3 showed the most homology,i.e., about 32% identity, with a Drosophila melanogaster BACR7A4.Ysequence (Accession # CAB51685). Comparison of SEQ ID NO:5 with nucleicacid sequences reported in GenBank indicates that SEQ ID NO:5 showed themost homology, i.e., about 39% identity, with a Rattus norvegicussynaptic vesicle protein 2B (SVP2B) mRNA (Accession # L10362). Percentidentity calculations were performed using SeqLab with defaultparameters.

Example 3

This Example describes the further characterization and isolation ofsynaptic vesicle 2B-like sequence nucleic acid molecules of the presentinvention.

A cDNA designated clone 2089-13 was isolated from the subtracted HMTlibrary as described in Example 1, the nucleic acid sequence of: cDNA'scoding strand is denoted herein as SEQ ID NO:7. DNA from clone 2089-13was purified, and the insert used for plaque hybridization screening ofthe unsubtracted HMT cDNA library as described in Example 2.Hybridization and plaque purification resulted in the isolation of aclone containing an about 3314 nucleotide synaptic vesicle 2B-likesequence, referred to herein as nCfSVP2₃₃₁₄ with a coding strand denotednucleic acid sequence SEQ ID NO:8 and a complementary sequence denotedSEQ ID NO:10. Translation of SEQ ID NO:8 suggests that nucleic acidmolecule nCrSVP2₃₃₁₄ encodes a full-length synaptic vesicle 2B-likeprotein of 773 amino acids, referred to herein as PCfSVP2₇₇₃, with anamino acid sequence denoted SEQ ID NO:9, assuming the initiation codonspans from nucleotide 302 through nucleotide 304 of SEQ ID NO:8 and thetermination codon spans from nucleotide 2621 through nucleotide 2623 ofSEQ ID NO:8. The coding region encoding PCfSVP2₇₇₃, is represented bynucleic acid molecule nCfSVP2₂₃₁₉, with a coding strand denoted SEQ IDNO:11 and a complementary strand denoted SEQ ID NO:12. The amino acidsequence of SEQ ID NO:9, predicts that PCfSVP2₇₇₃ has an estimatedmolecular weight of about 84.6 kDa and an pt of about 7.1.

Comparison of amino acid sequence SEQ ID NO:9 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:9 showed the most homology,i.e., about 68% identity, with a Drosophila melanogaster CG3168 geneproduct sequence (Accession # AAF46193). Comparison of SEQ ID NO:11 withnucleic acid sequences reported in GenBank indicates that SEQ ID NO:11showed the most homology, i.e., about 40% identity, with a Homo sapiensKIAA0735 sequence (Accession # NM014848). Percent identity calculationswere performed using SeqLab with default parameters.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

1. An isolated nucleic acid molecule selected from the group consistingof a flea cDNA molecule and a flea RNA molecule, wherein said nucleicacid molecule is at least 30 nucleotides in length and hybridizes with anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, andSEQ ID NO:12, under conditions comprising (a) hybridizing in a solutioncomprising 1×SSC in the absence of nucleic acid helix destabilizingcompounds, at a temperature of 37° C. and (b) washing in a solutioncomprising 1×SSC in the absence of helix destabilizing compounds, at atemperature of 47° C.
 2. The nucleic acid molecule of claim 1, whereinsaid nucleic acid molecule has a nucleic acid sequence that is at least70% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ IDNO:12, wherein percentage identity is determined using theNeedleman-Wunsch algorithm available in a SeqLab software program, usingSeqLab default parameters.
 3. The nucleic acid molecule of claim 1,wherein said nucleic acid molecule comprises a nucleic acid sequence atleast 30 nucleotides in length identical to a 30 nucleotide portion of anucleic acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
 4. The nucleic acidmolecule of claim 1, wherein said nucleic acid molecule is selected fromthe group consisting of: a nucleic acid molecule that encodes a proteinhaving an amino acid sequence selected from the group consisting of SEQID NO:3 and SEQ ID NO:9.
 5. A recombinant molecule comprising a nucleicacid molecule as set forth in claim 1 operatively linked to atranscription control sequence.
 6. A recombinant virus comprising anucleic acid molecule as set forth in claim
 1. 7. A recombinant cellcomprising a nucleic acid molecule as set forth in claim
 1. 8. Acomposition comprising an excipient and an isolated nucleic acidmolecule of claim
 1. 9. (Canceled)
 10. The composition of claim 8,further comprising a component selected from the group consisting of anadjuvant and a carrier.
 11. A method to produce a protein encoded by anisolated nucleic acid molecule of claim 1, said method comprisingculturing a cell transformed with a nucleic acid molecule encoding saidprotein.
 12. The method of claim 11, wherein said protein has an aminoacid sequence selected from the group consisting of: SEQ ID NO:3 and SEQID NO:9.
 13. The method of claim 11, wherein said nucleic acid moleculecomprises a nucleic acid sequence selected from the group consisting of:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, and/orSEQ ID NO:11.
 14. An isolated nucleic acid molecule having a nucleicacid sequence comprising at least 30 nucleotides identical in sequenceto a 30 nucleotide portion of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, andSEQ ID NO:12.
 15. An isolated protein encoded by a nucleic acid moleculeselected from the group consisting of: (a) a nucleic acid molecule atleast 30 nucleotides in length that hybridizes with a nucleic acidmolecule having a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, and/or SEQ IDNO:12, under conditions comprising (i) hybridizing in a solutioncomprising 1×SSC in the absence of helix destabilizing compounds, at atemperature of 37° C. and (ii) washing in a solution comprising 1×SSC inthe absence of helix destabilizing compounds, at a temperature of 47°C.; and (b) an isolated protein selected from the group consisting of:(1) a protein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:9; and (2) a protein comprisingan at least 10 contiguous amino acid portion identical in sequence to anat least 10 contiguous amino acid portion of an amino acid sequence of(1).
 16. The protein of claim 15, wherein said protein is encoded by anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7,SEQ ED NO:8, and/or SEQ ID NO:11.
 17. The protein of claim 15, whereinsaid protein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:9.
 18. A composition comprisingan excipient and an isolated protein of claim
 15. 19. (Canceled)
 20. Anisolated antibody that selectively binds to a protein as set forth inclaim
 15. 21. A composition comprising an excipient and an isolatedantibody of claim
 20. 22. (Canceled)
 23. A method to detect an inhibitorof flea synaptic vesicle activity, said method comprising (a) contactingan isolated flea synaptic vesicle protein of claim 15, with a putativeinhibitory compound under conditions in which, in the absence of saidcompound, said protein has flea synaptic vesicle protein activity, and(b) determining if said putative inhibitory compound inhibits fleasynaptic vesicle protein activity.
 24. (Canceled)
 25. (Canceled)